Use of cloprostenol and fluprostenol analogues to treat glaucoma and ocular hypertension

ABSTRACT

Disclosed is the use of cloprostenol and fluprostenol analogues and combinations thereof with other medicaments for the treatment of glaucoma and ocular hypertension and ophthalmic compositions therefor. Also disclosed are methods of treating optic nerve disorders using the cloprostenol and fluprostenol analogues.

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/917,795, filed Aug. 21, 1997, now U.S. Pat. No.5,889,052, which is a continuation of U.S. patent application Ser. No.08/769,293, filed Dec. 18, 1996, now U.S. Pat. No. 5,665,773, which is acontinuation of U.S. patent application Ser. No. 08/280,681, filed Jul.26, 1994, now abandoned, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/101,598 filed Aug. 3, 1993, now U.S. Pat. No.5,510,383.

BACKGROUND OF THE INVENTION

The present invention relates to the treatment of glaucoma and ocularhypertension. In particular, the present invention relates to the use ofcloprostenol and fluprostenol analogues for the treatment of glaucomaand ocular hypertension.

Cloprostenol and fluprostenol, both known compounds, are syntheticanalogues of PGF_(2α), a naturally-occurring F-series prostaglandin(PG). Structures for PGF_(2α)(I), cloprostenol (II), and fluprostenol(III), are shown below:

The chemical name for cloprostenol is16-(3-chlorophenoxy)-17,18,19,20-tetranor PGF_(2α). Monograph No. 2397(page 375) of The Merck Index, 11th Edition (1989) is incorporatedherein by reference to the extent that it describes the preparation andknown pharmacological profiles of cloprostenol. Fluprostenol has thechemical name 16-(3-trifluoromethylphenoxy)-17,18,19,20-tetranorPGF_(2α). Monograph No. 4121 (pages 656-657) of The Merck Index, 11thEdition (1989) is incorporated herein by reference to the extent that itdescribes the preparation and known pharmacological profiles offluprostenol. Cloprostenol and fluprostenol are 16-aryloxy PGs and, inaddition to the substituted aromatic ring, differ from the naturalproduct PGF_(2α) in that an oxygen atom is embedded within the lower(omega) chain. This oxygen interruption forms an ether functionality.

Naturally-occurring prostaglandins are known to lower intraocularpressure (lOP) after topical ocular instillation, but generally causeinflammation, as well as surface irritation characterized byconjunctival hyperemia and edema. Many synthetic prostaglandins havebeen observed to lower intraocular pressure, but such compounds alsoproduce the aforementioned side effects which severely restrict clinicalutility.

SUMMARY OF THE INVENTION

It has now been unexpectedly found that certain novel cloprostenol andfluprostenol analogues are useful in treating glaucoma and ocularhypertension. In particular, topical application of ophthalmiccompositions comprising these novel cloprostenol and fluprostenolanalogues result in significant lOP reduction.

DETAILED DESCRIPTION OF THE INVENTION

The compounds useful in the present invention have the following generalformula:

wherein:

R¹=H; C₁-C₁₂ straight-chain or branched alkyl; C₁-C₁₂ straight-chain orbranched acyl; C₃-C₈ cycloalkyl; or a cationic salt moiety;

R₂, R₃=H, or C₁-C₅ straight-chain or branched alkyl; or R₂ and R₃ takentogether may represent O;

X=O, S, or CH₂;

- - - represents any combination of a single bond, or a cis or transdouble bond for the alpha (upper) chain; and a single bond or transdouble bond for the omega (lower) chain;

R₉=H, C₁-C₁₀ straight-chain or branched alkyl, or C₁-C₁₀ straight-chainor branched acyl;

R₁₁=H, C₁-C₁₀ straight-chain or branched alkyl, or C₁-C₁₀ straight-chainor branched acyl;

Y=O; or H and OR₁₅ in either configuration wherein R₁₅=H, C₁-C10straight-chain or branched alkyl, or C₁-C₁₀ straight-chain or branchedacyl; and

Z=Cl or CF₃;

with the proviso that when R₂ and R₃ taken together represent O, thenR₁≠C₁-C₁₂ straight-chain or branched acyl; and when R₂=R₃=H, then R₁≠acationic salt moiety.

The term “acyl” represents a group that is linked by a carbon atom thathas a double bond to an oxygen atom and single bond to another carbonatom.

The term “acylamino” represents a group that is linked by an amino atomthat is connected to a carbon atom has a double bond to an oxygen groupand a single bond to a carbon atom or hydrogen atom.

The term “acyloxy” represents a group that is linked by an oxygen atomthat is connected to a carbon that has a double bond to an oxygen atomand single bond to another carbon atom.

The term “alkenyl” includes straight or branched chain hydrocarbongroups having 1 to 15 carbon atoms with at least one carbon-carbondouble bond. The chain hydrogens may be substituted with other groups,such as halogen. Preferred straight or branched alkeny groups include,allyl, 1-butenyl, 1-methyl-2-propenyl and 4-pentenyl.

The term “alkoxy” represents an alkyl group attached through an oxygenlinkage.

The term “alkyl” includes straight or branched chain aliphatichydrocarbon groups that are saturated and have 1 to 15 carbon atoms. Thealkyl groups may be substituted with other groups, such as halogen,hydroxyl or alkoxy. Preferred straight or branched alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl and t-butyl.

The term “alkylamino” represents an alkyl group attached through anitrogen linkage.

The term “alkynyl” includes straight or branched chain hydrocarbongroups having 1 to 15 carbon atoms with at least one carbon-carbontriple bond. The chain hydrogens may be substituted with other groups,such as halogen. Preferred straight or branched alkynyl groups include,2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl and 2-pentynyl.

The term “aryl” refers to carbon-based rings which are aromatic. Therings may be isolated, such as phenyl, or fused, such as naphthyl. Thering hydrogens may be substituted with other groups, such as loweralkyl, or halogen.

The term “carbonyl” represents a group that has a carbon atom that has adouble bond to an oxygen atom.

The term “carbonylalkoxy” represents a group that is linked by a carbonatom that has a double bond to an oxygen atom and a single bond to analkoxy group.

The term “cationic salt moiety” includes alkali and alkaline earth metalsalts as well as ammonium salts.

The term “carbonyloxyl” represents a group that is linked by a carbonatom that has a double bond to an oxygen atom and a single bond to asecond oxygen atom.

The term “cycloalkyl” includes straight or branched chain, saturated orunsaturated aliphatic hydrocarbon groups which connect to form one ormore rings, which can be fused or isolated. The rings may be substitutedwith other groups, such as halogen, hydroxyl or lower alkyl. Preferredcycloalkyl groups include cyclopropyl, cyclobutyl, cylopentyl andcyclohexyl.

The term “dialkylamino” represents two alkyl groups attached through anitrogen linkage.

The term “halogen” and “halo” represents fluoro, chloro, bromo, or iodo.

The term “heteroaryl” refers to aromatic hydrocarbon rings which containat least one heteroatom such as O, S, or N in the ring. Heteroaryl ringsmay be isolated, with 5 to 6 ring atoms, or fused, with 8 to 10 atoms.The heteroaryl ring(s) hydrogens or heteroatoms with open valency may besubstituted with other groups, such as lower alkyl or halogen. Examplesof heteroaryl groups include imidazole, pyridine, indole, quinoline,furan, thiophene, pyrrole, tetrahydroquinoline, dihydrobenzofuran, anddihydrobenzindole.

The term “lower alkyl” represents alkyl groups containing one to sixcarbons (C₁-C₆).

Preferred compounds include: cloprostenol isopropyl ester (Table 1,compound 1A), fluprostenol isopropyl ester (compound 1B),16-phenoxy-17,18,19,20-tetranor PGF_(2α) isopropyl ester (compound 2),17-phenyl-18,19,20-trinor PGF_(2α) isopropyl ester (compound 3),13,14-dihydro-17-phenyl-18,19,20-trinor PGF_(2α)) isopropyl ester(compound 4), the 3-oxa form of cloprostenol isopropyl ester (Table 2,compound 5), 13,14-dihydrofluprostenol isopropyl ester (compound 6),cloprostenol-1-ol (compound 7), and 13,14-dihydrocloprostenol-1-olpivaloate (compound 8).

TABLE 1 COMPOUND NAME COMPOUND STRUCTURE 1A Cloprostenol, isopropylester

1B Fluprostenol, isopropyl ester

2 16-Phenoxy-17,18,19,20- tetranor PGF_(2α), isopropyl ester

3 17-Phenyl-18,19,20-trinor PGF_(2α), isopropyl ester

4 13,14-Dihydro-17-phenyl- 18,19,20-trinor PGF_(2α), isopropyl ester

TABLE 2 COMPOUND NAME COMPOUND STRUCTURE 5 3-oxacloprostenol isopropylester

6 13,14-dihydrofluprostenol isopropyl ester

7 cloprostenol-1-ol

8 13,14-dihydrocloprostenol-1-ol pivaloate

The compounds of formula (IV) are useful in lowering intraocularpressure and thus are useful in the treatment of glaucoma. Suchcompounds are also useful in improving optic nerve head blood flow andthe treatment of optic nerve disorders (including without limitationretarding visual field loss and improving visual acuity), the latterbeing generally described in U.S. Pat. No. 5,773,471, the contents ofwhich are by this reference incorporated herein. It is furthercontemplated that the compounds of the present inventions can be usedwith other medicaments known to be useful in the treatment of glaucomaor ocular hypertension, either separately or in combination. Forexample, the prostaglandin analogs of the present invention can becombined with (i) beta-blockers, such as timolol, betaxolol, levobunololand the like (see U.S. Pat. No. 4,952,581); (ii) carbonic anhydraseinhibitors, such as brinzolamide; (iii) adrenergic agonists includingclonidine derivatives, such as apraclonidine or brimonidine (see U.S.Pat. No. 5,811,443); and (iv) cholinergic agonists, such as pilocarpine.The disclosures of U.S. Pat. Nos. 4,952,581 and 5,811,443 areincorporated herein by this reference. The preferred route ofadministration is topical. The dosage range for topical administrationis generally between about 0.01 and about 1000 micrograms per eye(μg/eye), preferably between about 0.1 and about 100 μg/eye, and mostpreferably between about 1 and 10 μg/eye. The compounds of the presentinvention can be administered as solutions, suspensions, or emulsions(dispersions) in a suitable ophthalmic vehicle.

In forming compositions for topical administration, the compounds of thepresent invention are generally formulated as between about 0.00003 toabout 3 percent by weight (wt %) solutions in water at a pH between 4.5to 8.0. The compounds are preferably formulated as between about 0.0003to about 0.3 wt % and, most preferably, between about 0.003 and about0.03 wt %. While the precise regimen is left to the discretion of theclinician, it is recommended that the resulting solution be topicallyapplied by placing one drop in each eye one or two times a day.

Other ingredients which may be desirable to use in the ophthalmicpreparations of the present invention include preservatives, co-solventsand viscosity building agents.

Antimicrobial Preservatives

Ophthalmic products are typically packaged in multidose form, whichgenerally require the addition of preservatives to prevent microbialcontamination during use. Suitable preservatives include: benzalkoniumchloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben,phenylethyl alcohol, edetate disodium, sorbic acid, ONAMER M®, or otheragents known to those skilled in the art. Such preservatives aretypically employed at a concentration between about 0.001% and about1.0% by weight.

Co-Solvents

Prostaglandins, and particularly ester derivatives, typically havelimited solubility in water and therefore may require a surfactant orother appropriate co-solvent in the composition. Such co-solventsinclude: Polysorbate 20, 60 and 80; Pluronic F-68, F-84 and P-103;Tyloxapol®; Cremophor® EL; sodium dodecyl sulfate; glycerol; PEG 400;propylene glycol; cyclodextrins; or other agents known to those skilledin the art. Such co-solvents are typically employed at a concentrationbetween about 0.01% and about 2% by weight.

Viscosity Agents

Viscosity greater than that of simple aqueous solutions may be desirableto increase ocular absorption of the active compound, to decreasevariability in dispensing the formulations, to decrease physicalseparation of components of a suspension or emulsion of formulationand/or otherwise to improve the ophthalmic formulation. Such viscositybuilding agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose or other agents known to those skilled in the art. Such agentsare typically employed at a concentration between about 0.01% and about2% by weight.

The following Examples 1-4 describe the synthesis of compounds 5-8(Table 2). These syntheses are representative in nature and are notintended to be limiting. Other compounds of formula (IV) may be preparedusing analogous techniques known to those skilled in the art.

In the examples below, the following standard abbreviations are used:g=grams (mg=milligrams); mol=moles (mmol=millimoles); mol %=molepercent; mL=milliliters; mm Hg=millimeters of mercury; mp=melting point;bp=boiling point; h=hours; and min=minutes. In addition, “NMR” refers tonuclear magnetic resonance spectroscopy and “Cl MS” refers to chemicalionization mass spectrometry.

EXAMPLE 1 Synthesis of 3-Oxacloprostenol (5)

A: Ethyl (3-chlorophenoxy)acetate (10)

Acetone (320 ml), 75 g (450 mmol) of ethyl bromoacetate, and 40.0 g (310mmol) of 3-chlorophenol were mixed together, then 69.8 g (505 mmol) ofpotassium carbonate was added. The mixture was mechanically stirred andheated to reflux for 4 h, and after cooling to room temperature, waspoured into 350 mL of ethyl acetate. To this was then cautiously added400 mL of 1M HCl, taking care to avoid excess foaming. The layers wereseparated and the aqueous layer was extracted with portions of ethylacetate (3×200 mL). The combined organic layers were dried over MgSO₄,filtered, concentrated, and the resulting solid was recrystallized fromhexane to afford 58 g (87%) of 10 as a white solid, m.p.=39-40° C. ¹HNMR δ 7.20-7.08 (m, 1 H), 6.95-6.82 (m, 2 H), 6.75-6.70 (m, 1 H), 4.53(s, 2 H), 4.21 (q, J=7.2 Hz, 2 H), 1.23 (t, J=7.2 Hz, 3 H).

B: Dimethyl [3-(3-chlorophenoxy)-2-oxoprop-1-yl]phosphonate (11)

To 20.6 g (166 mmol, 238 mol %) of dimethyl methylphosphonate in 110 mLof THF at −78° C. was added dropwise 65 mL (162 mmol, 232 mol %) of a2.5M solution of n-BuLi in hexanes. After addition was complete, themixture was stirred for an additional 1 h, after which 15.0 g (69.9mmol) of aryloxyester 10 in 40 mL of THF was added dropwise. Thereaction was stirred for 1 h and then quenched by the addition of 100 mLof saturated NH₄Cl. The mixture was poured into 200 mL of a 1/1 mixtureof saturated NaCl/ethyl acetate, layers were separated, and the aqueouslayer was further extracted with ethyl acetate (2×100 mL). Combinedorganic layers were dried over MgSO₄, filtered, and concentrated, toafford 20.5 g (100%) of 11 as a viscous oil. ¹H NMR δ 7.22 (t, J=8.1 Hz,1 H), 7.05-6.90 (m, 2 H), 6.85-6.78 (m, 1 H), 4.72 (s, 2 H), 3.84 (s, 3H), 3.78 (s, 3 H), 3.27 (d, J=22.8 Hz, 2 H).

C: (3aR, 4R, 5R,6aS)-5-(Benzoyloxy)-4-[(E)-4-(3-chlorophenoxy)-3-oxo-1-butenyl]-hexahydro-2H-cyclopenta[b]furan-2-one(13)

Phosphonate 11 (20.5 g, 70.0 mmol), 2.6 g (62 mmol) of LiCl, and 200 mLof THF were mixed together at 0° C. and 6.10 g (60.4 mmol) of NEt₃ wasadded. Aldehyde 12 (14.0 g, 51.1 mmol) dissolved in 50 mL of CH₂Cl₂ wasthen added dropwise. After 1 h, the reaction was poured into 200 mL of a1/1 mixture of saturated NH₄Cl/ethyl acetate, the layers were separated,and the aqueous layer was extracted with ethyl acetate (2×100 mL).Combined organic layers were dried over MgSO₄, filtered, concentrated,and the residue was chromatographed on silica gel eluting with ethylacetate/hexanes, 3/2, to afford 16.2 g (72%) of 13 as a whitecrystalline solid, m.p.=101.0-102.0° C. ¹H NMR δ 8.0-7.9 (m, 2 H),7.62-7.52 (m, 1 H), 7.50-7.38 (m, 2 H), 7.18 (t, J=8.2 Hz, 1 H),7.0-6.82 (m, 3 H), 6.75-6.70 (m, 1 H), 6.54 (d, J=15.1 Hz, 1 H), 5.32(q, J=6.2 Hz, 1 H), 5.12-5.05 (m, 1 H), 4.66 (s, 2 H), 3.0-2.8 (m, 3 H),2.7-2.2 (m, 3 H).

D: (3aR, 4R, 5R,6aS)-5-(Benzoyloxy)-4-[(E)-(3R)4-(3-chlorophenoxy)-3-hydroxy-1-butenyl]-hexahydro-2H-cyclopenta[b]furan-2-one(14)

To a solution of 9.70 g (22.0 mmol) of enone 13 in 60 mL of THF at −23°C. was added dropwise a solution of 11.1 g (34.6 mmol) of(−)-B-chlorodiisopinocampheylborane in 30 mL of THF. After 4 h, thereaction was quenched by the dropwise addition of 5 mL of methanol andthen warmed to room temperature. After pouring into 200 mL of a 2/1mixture of ethyl acetate/saturated NH₄Cl, the layers were separated, andthe aqueous phase was extracted with ethyl acetate (2×100 mL). Combinedorganic layers were dried over MgSO₄, filtered, concentrated, and theresidue was chromatographed on silica gel eluting with ethylacetate/hexanes, 3/2, to afford 4.7 g (48%) of 14 as a white solid, m.p.101.0-102.5° C. ¹H NMR δ 8.05-7.95 (m, 2 H), 7.62-7.40 (m, 3 H), 7.18(t, J=8.0 Hz, 1 H), 7.0-6.92 (m, 1 H), 6.85 (t, J=2.1 Hz, 1 H),6.77-6.70 (m, 1 H), 5.85 (d of d, J=6.2, 15.5 Hz, 1 H), 5.72 (d of d,J=4.5, 15.5 Hz, 1 H), 5.30 (q, J=5.8 Hz, 1 H), 5.12-5.04 (m, 1 H),4.58-4.48 (m, 1 H), 3.92 (d of d, J=3.5, 9.3 Hz, 1 H), 3.80 (d of d,J=7.3, 9.4 Hz, 1 H), 2.9-2.2 (m, 8 H).

E: (3aR, 4R, 5R,6aS)-4-[(E)-(3R)-4-(3-Chlorophenoxy)-3-(tetrahydropyran-2-yloxy)-1-butenyl]-5-(tetrahydropyran-2-yloxy)-hexahydro-2H-cyclopenta[b]furan-2-one(16)

To a mixture of 5.1 g (11.5 mmol) of 14 in 200 mL of methanol was added1.7 g (12 mmol) of K₂CO₃. After 1 h, the mixture was poured into 100 mLof 0.5M HCl and extracted with ethyl acetate (3×100 mL). The combinedorganic layers were washed successively with water (2×100 mL) andsaturated NaCl (2×100 mL). The organic layer was dried over MgSO₄,filtered, and concentrated to afford 4.85 g of crude diol 15, which wasused in the next step without further purification.

To a mixture of 4.85 g of crude 15 and 2.4 g (28 mmol) of3,4-dihydro-2H-pyran in 75 mL of CH₂Cl₂ at 0° C. was added 370 mg (1.9mmol) of p-toluenesulfonic acid monohydrate. After stirring for 45 min,the reaction was poured into 40 mL of saturated NaHCO₃, layers wereseparated, and the aqueous layer was extracted with CH₂Cl₂ (2×40 mL).The combined organic layers were dried over MgSO₄, filtered, andconcentrated. The residue was chromatographed on silica gel eluting with40% ethyl acetate in hexanes, to afford 6.0 g (100%) of 16 as an oil. ¹HNMR (CDCl₃) δ (characteristic peaks only) 7.25-7.14 (m, 1 H), 6.95-6.87(m, 2 H), 6.83-6.72 (m, 1 H), 5.8-5.4 (m, 4 H), 5.1-4.8 (m, 2 H).

F: (13E)-(9S, 11R,15R)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-2,3,4,5,6,17,18,19,20-nonanor-9-triethylsilyloxy-13-prostenolTriethylsilyl Ether (18)

To a suspension of 400 mg (10.5 mmol) of lithium aluminum hydride in 20mL of THF at 0° C. was added dropwise a solution of 4.5 g (8.8 mmol) oflactone 16 in 20 mL of THF. After 1 h at 0° C. the mixture wascautiously poured into 100 mL of a 1/1 mixture of ice-cold saturatedNH₄Cl/ethyl acetate. The layers were separated, and the aqueous layerwas extracted with ethyl acetate (2×50 mL). The combined organic layerswere dried over MgSO₄, filtered, and concentrated to afford 4.5 g (100%)of diol 17 which was used in the next step without further purification.

Triethylsilyl chloride (3.0 g, 20 mmol) was added to a mixture of 4.5 g(8.8 mmol) of crude 17, 40 mL of DMF, 1.85 g (27.0 mmol) of imidazole,and 310 mg (2.5 mmol) of 4-(dimethylamino)pyridine. After 2 h, thereaction was poured into 100 mL of a 1/1 mixture of ethylacetate/saturated NH₄Cl, layers were separated, and the aqueous layerwas extracted with ethyl acetate (2×25 mL). The combined organic layerswere washed with water (3×25 mL), dried over MgSO₄, and concentrated.The residue was chromatographed on silica gel eluting with 20% ethylacetate in hexane to afford 5.2 g (80%) of 18. ¹H NMR (CDCl₃) δ(characteristic peaks only) 7.22-7.12 (m, 1 H), 6.95-6.88 (m, 2 H),6.83-6.71 (m, 1 H), 5.8-5.4 (m, 4 H), 5.1-4.8 (m, 2 H), 1.0-0.85 (m, 18H), 0.7-0.5 (m, 12 H).

G: (13E)-(9S, 11R,15R)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-2,3,4,5,6,17,18,19,20-nonanor-9-triethylsilyloxy-13-prostenal(19)

To a mixture of 1.6 g (12.6 mmol) of oxalyl chloride and 15 mL of CH₂Cl₂at −78° C. was added dropwise a solution of 1.54 g (19.7 mmol) of DMSOin 2 mL of CH₂Cl₂. After 10 min, 4.6 g (6.2 mmol) of bissilane 18 in 8mL of CH₂Cl₂ was added dropwise. After 95 min, 3.0 g (30 mmol) of NEt₃was added. The mixture was then warmed to room temperature and pouredinto 70 mL of saturated NH₄Cl. The solution was extracted with of CH₂Cl₂( 3×70 mL) and the combined organic layers were dried over MgSO₄,filtered, and concentrated. The residue was chromatographed on silicagel eluting with 20% ethyl acetate in hexane to afford 2.06 g (53%) of19 as well as 1.5 g (26%) recovered 18. ¹H NMR (CDCl₃) δ (characteristicpeaks only) 9.78 (t, J=1.4 Hz, 1 H), 7.22-7.12 (m, 1 H), 6.95-6.88 (m, 2H), 6.83-6.71 (m, 1 H), 5.8-5.4 (m, 4 H) 5.1-4.8 (m, 2 H), 1.0-0.85 (m,18 H), 0.7-0.5 (m, 12 H).

H: (5Z, 13E)-(9S, 11R,15R)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-2,3,4.17,18,19,20-heptanor-9-triethylsilyloxy-5,13-prostadienoicAcid Methyl Ester (21)

To a solution of 1.35 g (4.24 mmol) of phosphonate 20 and 2.60 g (9.84mmol) of 18-crown-6 in 20 mL of THF at −78° C. was added dropwise 6.9 mL(3.45 mmol) of a 0.5M solution of potassium hexamethyldisilazane intoluene. After stirring for 15 min, a solution of 1.65 g (2.64 mmol) ofaldehyde 19 in 20 mL of THF was added dropwise. One hour later, themixture was poured into 100 mL of saturated NH₄Cl/ethyl acetate, 1/1,layers were separated, and the aqueous layer was extracted with ethylacetate (3×30 mL). The combined organic layers were dried over MgSO₄,filtered, concentrated and the residue was chromatographed on silica geleluting with 20% ethyl acetate in hexane to afford 1.135 g (63%) of 21.¹H NMR (CDCl₃) δ (characteristic peaks only) 7.22-7.11 (m, 1 H),6.97-6.86 (m, 2 H), 6.85-6.75 (m, 1 H), 6.4-6.2 (m, 1 H), 5.8-5.32 (m, 3H), 3.66 (s, 3 H).

I: (5Z, 13E)-(9S, 11R,15R)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-2,3,4,17,18,19,20-heptanor-9-triethylsilyloxy-5,13-prostadien-1-ol(22)

To a solution of 850 mg (1.25 mmol) of ester 21 in 10 mL of THF at 0° C.was added 2.4 mL (3.6 mmol) of a 1.5M solution of diisobutylaluminumhydride in toluene. After 1 h, the mixture was poured into 20 mL ofsaturated NH₄Cl and was extracted with ethyl acetate (3×20 mL). Combinedorganic layers were dried over MgSO₄, filtered, and concentrated down to800 mg (98%) of 22 as an oil. ¹H NMR (CDCl₃) δ (characteristic peaksonly) 7.25-7.15 (m, 1 H), 6.97-6.90 (m, 2 H), 6.86-6.75 (m, 1 H),5.81-5.41 (m, 4 H).

J: (5Z, 13E)-(9S, 11R, 15R)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-3-oxa-17,18,19,20-tetranor-9-triethylsilyloxy-5,13-prostadienoicAcid Isopropyl Ester (23)

To a solution of 415 mg (6.37 mmol) of alcohol 22 in 4 mL of THF at −78°C. was added dropwise 0.35 mL (0.87 mol) of a 2.5M solution of n-BuLi inhexane. After 15 min, this solution was transferred via syringe to a−78° C. solution of 195 mg (1.08 mmol) of isopropyl bromoacetate in 2 mLof THF. The mixture was kept at −78° C. for 40 min, warmed to roomtemperature overnight, and then poured into 20 mL of a 1/1 mixture ofsaturated NH₄Cl/ethyl acetate. Layers were separated, and the aqueouslayer was extracted with ethyl acetate ( 2×10 mL). The combined organiclayers were dried over MgSO₄, filtered, concentrated, and the residuewas chromatographed on silica gel (20% ethyl acetate in hexane) toafford 242 mg (53%) of 23 as an oil. ¹H NMR (CDCl₃) δ (characteristicpeaks only) 7.24-7.15 (m,1 H), 6.97-6.90 (m, 2 H), 6.86-6.75 (m, 1 H),5.81-5.41 (m, 4 H), 1.57 (d, J=5.7Hz, 6 H).

K: (5Z, 13E)-(9S, 11R, 15R)-16-(3-Chlorophenoxy)-3-oxa-17,18,19,20-tetranor-9,11,15-trihydroxy-5,13-prostadienoicAcid Isopropyl Ester (5)

To a solution of 230 mg (0.32 mmol) of silane 23 in 5 mL of THF at roomtemperature was added 0.33 mL (0.33 mmol) of a 1M solution of Bu₄NF inTHF. After 20 min, the reaction was poured into 4 mL of saturated NH₄Cland was extracted with ethyl acetate (4×5 mL). The combined organiclayers were dried over MgSO₄, filtered, concentrated, and the residuewas chromatographed on silica gel (ethyl acetate/hexane, 1/1), to afford126 mg (65%) of desilylated compound 24.

To 120 mg of 24 in 5 mL of methanol was added 0.4 mL of 2M HCl. After 1h, the mixture was added to 3 mL of saturated NaHCO₃, and the resultingmixture was extracted with ethyl acetate (3×8 mL). Combined organiclayers were dried over MgSO₄, filtered, concentrated. The resultingresidue was then chromatographed on silica gel eluting with ethylacetate to afford 54 mg (56%) of 5. ¹³C NMR (CDCl₃) δ 169.92 (C), 159.26(C), 135,13 (CH), 134.95 (CH), 134.81 (C), 124.93 (CH), 121.22 (CH),115.06 (CH), 113.08 (CH), 77.75 (CH), 72.02 (CH), 71.94 (CH₂), 70.76(CH₂), 68.77 (CH), 67.78 (CH₂), 66.50 (CH₂), 55.46 (CH), 49.93 (CH),42.47 (CH₂), 25.85 (CH₂), 21.75 (CH₃). Cl MS, m/z calcd. for C₂₄H₃₄O₇Cl₁(MH⁺), 469.1993, found 469.1993.

EXAMPLE 2 Synthesis of 13,14-Dihydrofluprostenol Isopropyl Ester

A: (3aR, 4R, R,6aS)-5-Hydroxy-4-[(3R)-4-(3-trifluoromethylphenoxy)-3-hydroxy-1-butyl]-hexahydro-2H-cyclopenta[b]furan-2-one(26)

A mixture of 1.2 g (3.2 mmol) of diol 25 (for synthesis of diol 25, seeU.S. Pat. No. 4,321,275 ) and 0.05 g of 10% (wt/wt) Pd/C in 20 mL ofmethanol was hydrogenated at 30 psi for 1.5 hours. After filtrationthrough a short pad of Celite® concentration afforded 1.2 g (100%) of 26as a colorless oil. ¹H NMR (CDCl₃) δ 7.44 (m, 2 H), 7.12 (m, 2 H), 4.95(dt, 1 H), 4.15-3.80 (m, 4 H), 2.82 (dd, J=10.8, 1 H), 2.55 (m, 2 H),2.3 (m, 1 H), 2.1-1.3 (m, 6 H).

B: (3aR, 4R, 5R,6aS)-5-(Tetrahydropyran-2-yloxy)-4-[(3R)-4-(3-trifluoromethylphenoxy)-3-(tetrahydropyran-2-yloxy)-1-butyl]-hexahydro-2H-cyclopenta[b]furan-2-one(27)

A mixture of 1.2 g (3.2 mmol) of diol 26 and 0.05 g of p-toluenesulfonicacid monohydrate in 100 mL of CH₂Cl₂ at 0° C. was treated with3,4-dihydro-2H-pyran (1.1 ml, 12 mmol) and the solution was stirred for2 h at 0° C. After pouring into saturated NaHCO₃, phases were separatedand the organic layer was dried over MgSO₄, filtered, concentrated, andpurified by chromatography on silica gel (1/1, hexanes EtOAc) to afford1.1 g of 27 as a clear, colorless oil. ¹H NMR (CDCl₃) δ 8.04 (dd, J=7.0,1.6, 1 H), 7.44 (m, 2 H), 7.12 (m, 1 H), 4.95 (dt, 1 H), 4.8 (m, 1 H),4.7 (m, 2 H), 4.15-3.80 (m, 4 H), 3.5 (m, 2 H), 2.82 (dd, J=10.8, 1 H),2.55 (m, 2 H), 2.3 (m, 1 H), 2.1-1.3 (m, 6 H).

C: (5Z)-(9S, 11R,15R)-11,15-Bis(tetrahydropyran-2-yloxy)-9-hydroxy-17,18,19,20-tetranor-16-(3-trifluoromethylphenoxy)-5-prostenoicAcid Isopropyl Ester (31)

To a solution of 2.1 g (3.9 mmol) of 27 in 100 mL of THF at −78° C. wasadded 3.9 mL (5.8 mmol) of a 1.5M solution of diisobutyaluminum hydridein toluene. The solution was stirred for 2 h, the n quenched by thesequential addition of 0.4 mL of isopropanol at −78° C. followed by 0.4mL of water at 23° C. Volatiles were removed under reduced pressure andthe aqueous solution was extracted with Et₂O/EtOAc (1/1). Organicextracts were dried over MgSO₄, filtered, and concentrated to furnish1.9 g of lactol 28.

To a 250 mL 3-necked round bottom flask equipped with a mechanicalstirrer and a thermometer were added anhydrous DMSO (100 mL) and NaH(80% dispersion in mineral oil; 0.48 g,16 mmol). The mixture was heatedto 75° C. (internal) for 30 min, after which it was allowed to cool toroom temperature for 1 h. Phosphonium bromide 29 (3.5 g, 8 mmol) wasthen added. After stirring for 30 minutes, 1.9 g (3.5 mmol) of lactol 28in 50 mL of DMSO was added, and the resulting solution was heated to 50°C. for 2 h and then brought to room temperature for 16 h. The solutionwas poured into 100 mL of water and approximately 2 mL of 50% NaOHadded. The aqueous phase was extracted with ether (3×100 mL), then madeacidic (pH=5.5) by the addition of a 10% citric acid solution, andextracted with Et₂O/hexanes, 2/1 (3×100 mL). The combined organicextracts were dried over MgSO₄, filtered, and concentrated to afford 1.9g of 30 as a colorless oil.

To 1.9 g of carboxylic acid 30 dissolved in 10 mL acetone was added 0.95g (6.0 mmol) of DBU and 1.0 g (6.1 mmol) of isopropyl iodide at 23° C.After 16 h, the solution was poured into 100 mL of water and extractedwith 100 mL of EtOAc. The organic extract was dried over MgSO₄,filtered, concentrated, and purified by silica gel chromatography (3/2,hexanes/EtOAc) to afford 1.9 g of isopropyl ester 31 as a colorless oil.¹H NMR (CDCl₃) δ 7.44 (t, 1 H), 7.12 (d, 1 H), 7.12 (dd, 2 H), 5.5-5.3(m, 2 H), 4.99 (heptet, 1 H), 4.15-3.80 (m, 4 H), 2.82 (dd, J=10.8, 1H), 2.55 (m, 2 H), 2.3 (m, 1 H), 2.1-1.3 (m, 24 H), 1.23 (s, 3 H), 1.20(s, 3 H).

D: (5Z)-(9S, 11R,15R)-17,18,19,20-Tetranor-16-(3-trifluoromethylphenoxy)-9,11,15-trihydroxy-5-prostenoicAcid Isopropyl Ester (6)

Ester 31 (1.9 g, 2.8 mmol) was dissolved in 14 mL of a mixture ofAcOH/THF/H₂O (4/2/1) and the solution was heated to 50° C. for 1 h,allowed to cool to 23° C., poured into a saturated solution of NaHCO₃,and extracted with Et₂O (2×100 mL) and EtOAc (100 mL). The combinedorganic extracts were dried over MgSO₄, filtered, concentrated, andpurified by silica gel chromatography (1/1, hexanes/EtOAc) to furnish0.5 g of triol 6 as a clear, colorless oil. ¹H NMR (CDCl₃) δ 7.44 (t,J=7.8, 1 H), 7.12 (dd, J=7.8, 2.0, 1 H), 7.12 (ddd, J=15.6, 7.2, 2.0, 2H), 5.5-5.3 (m, 2 H), 4.99 (heptet, J=6.3, 1 H), 4.15-3.80 (m, 4 H), 3.2(d, 1 H), 2.95 (s, 1 H), 2.82 (dd, J=10.8, 1 H), 2.75 (d, J=5.9, 1 H),2.55 (m, 2 H), 2.3 (m, 1 H), 2.1-1.3 (m, 24 H), 1.23 (s, 3 H), 1.20 (s,3 H). ¹³C NMR (CDCl₃) δ 173.5, 158.7, 132.1, 131.5, 130.0, 129.5, 129.2,123.3, 120.8, 117.7, 117.6, 111.4, 111.4, 78.6, 74.4, 72.4, 69.9, 67.6,52.6, 51.7, 42.5, 34.0, 31.5, 29.4, 26.8, 26.6, 24.9, 21.7.

EXAMPLE 3 Synthesis of Cloprostenol-1-ol (7)

A: (5Z, 13E)-(9S, 11R,15R)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-9-hydroxy-17,18,19,20-tetranor-5,13-prostadienoicAcid Isopropyl Ester (34)

A 1.5M solution of diisobutylaluminum hydride in toluene (10 mL, 15mmol) was added dropwise to a solution of 5.8 g (11.4 mmol) of lactone16 in 55 mL of THF at −78° C. After 1 h, 10 mL of methanol was addeddropwise, and the mixture was stirred for 10 min at −78° C. before beingwarmed to room temperature. The mixture was then poured into 100 mL of a1/1 solution of saturated aqueous potassium sodium tartrate/ethylacetate and stirred. After separating layers, the aqueous phase wasextracted with ethyl acetate (2×40 mL). Combined organic layers weredried over MgSO₄, filtered, concentrated, and purified by silica gelchromatography (3/2, ethyl acetate/hexane), to afford 4.4 g (76%) oflactol 33, which was used immediately in the next step.

A 1M solution of potassium t-butoxide in THF (50.0 ml) was addeddropwise to 12.1 g (27.3 mmol) of phosphonium salt 29 in 100 mL of THFat 0° C. After 30 min, a solution of 4.4 g (8.6 mmol) of lactol 33 in 20mL of THF was added dropwise, and the mixture was stirred at roomtemperature overnight. The solution was then poured into 150 mL of a 1/1mixture of ethyl acetate/saturated NH₄Cl. Layers were separated and theaqueous layer was extracted with ethyl acetate (2×100 mL). Combinedorganic layers were dried over MgSO₄, filtered, concentrated, and theresidue was redissolved in 80 mL of acetone. To this was added 6.5 g (45mmol) of DBU followed by 7.3 g (43 mmol) of isopropyl iodide. Afterstirring overnight, the reaction was poured into 100 mL of a 1/1 mixtureof ethyl acetate/saturated NH₄Cl. Layers were then separated and theaqueous phase was further extracted with ethyl acetate (2×100 mL). Thecombined organic layers were dried over MgSO₄, filtered, concentrated,and purified by silica gel chromatography (40% ethyl acetate in hexane)to afford 2.92 g (53% from lactone 16) of ester 34.

B: (5Z, 13E)-(9S, 11R,15R)-16-(3-Chlorophenoxy)-17,18,19,20-tetranor-9,11,15-trihydroxy-5,13-prostadienol(7)

A solution of 500 mg (0.79 mmol) of 34 in 10 mL of THF was addeddropwise to 61 mg (1.60 mmol) of lithium aluminum hydride in 20 mL ofTHF at 0° C. After 40 min, the reaction was carefully poured into 15 mLof saturated NH₄Cl, and the mixture was then extracted with ethylacetate ( 3×40 mL). Combined organic layers were dried over MgSO₄,filtered, and concentrated to afford 500 mg of crude 35.

To a solution of 500 mg of 35 in 20 mL of methanol was added 0.5 mL of2M HCl. After 1 h, the reaction was quenched with 20 mL of saturatedNaHCO₃ and the mixture was extracted with ethyl acetate (4×30 mL). Thecombined organic layers were dried over MgSO₄, filtered, andconcentrated. Silica gel chromatography (EtOAc) provided 101 mg (31%from 34) of 7. ¹³C NMR (CDCl₃) δ 159.27 (C), 135.44 (CH), 134.82 (C),130.64 (CH), 130.26 (CH), 128.23 (CH), 121.25 (CH), 115.07 (CH), 113.08(CH), 77.35 (CH), 72.35 (CH), 71.90 (CH₂),70.89 (CH), 62.22 (CH₂), 55.40(CH), 49.87 (CH), 42.79 (CH₂), 31.83 (CH₂), 26.77 (CH₂), 25.60 (CH₂),25.33 (CH₂). Cl MS m/z calcd for C₂₂H₃₂O₅Cl₁ (MH+) 411.1938, found411.1938.

EXAMPLE 4 Synthesis of 13,14-Dihydrocloprostenol-1-ol Pivaloate (8)

A: (3aR, 4R, 5R,6aS)4-[(3R)-4-(3-Chlorophenoxy)-3-hydroxybutyl]-5-hydroxy-hexahydro-2H-cyclopenta[b]furan-2-one(37)

A mixture of 2.4 g (5.4 mmol) of 14 and 250 mg of 10% (wt/wt) Pd/C in 35mL of ethyl acetate was hydrogenated at 40 psi for 1 h. After filtrationthrough a short pad of Celite®, the filtrate was evaporated down to 2.3g (100%) of hydrogenated product 36.

The crude benzoate 36 was dissolved in 25 mL of methanol, and 610 mg(4.4 mmol) of K₂CO₃ was added. After 3.5 h, the mixture was poured into100 mL of water/ethyl acetate (1/1). Layers were separated, and theaqueous phase was further extracted with ethyl acetate (2×50 mL). Thecombined organic layers were dried over MgSO₄, filtered andconcentrated. Silica gel chromatography (EtoAc) provided 1.50 g (82%) of37 as a white solid, m.p.=102.0-103.5° C. ¹H NMR δ 7.22 (t, J=8.2 Hz, 1H), 7.0-6.94 (m, 1 H), 6.91-6.88 (t, J=2.1 Hz, 1 H), 6.83-6.77 (m, 1 H),4.97 (dt, J=3.0, 8.3 Hz, 1 H), 4.12-3.91 (m, 3 H), 3.82 (dd, J =7.4, 9.0Hz, 1 H), 2.85 (dd, J=8.0, 16.5 Hz, 1H), 2.6-1.4 (m, 11 H).

B: (3aR, 4R, 5R,6aS)-4-[(3R)-4-(3-Chlorophenoxy)-3-(tetrahydropyran-2-yloxy)butyl]-5-(tetrahydropyran-2-yloxy)-hexahydro-2H-cyclopenta[b]furan-2-one(38)

Diol 37 (3.4 g, 10 mmol) and 2.2 g (26 mmol) of 3,4-dihydro-2H-pyranwere dissolved in 80 mL of CH₂Cl₂, and 240 mg (1.3 mmol) ofp-toluenesulfonic acid monohydrate was added at 0° C. After 1 h, thereaction was poured into 50 mL of saturated NaHCO₃ and the mixture wasextracted with CH₂Cl₂ (3×40 mL). The combined organic layers were driedover MgSO₄, filtered, concentrated, and the residue was chromatographedon silica gel (hexane/ethyl acetate, 1/1) to afford 4.5 g (87%) ofbis-THP ether 38.

C: (5Z)-(9S, 11R,15R)-11,15-Bis(tetrahydropyran-2-yloxy)-16-(3-chlorophenoxy)-9-hydroxy-17,18,19,20-tetranor-5-prostenoicAcid Isopropyl Ester (41)

A 1.5M solution of diisobutylaluminum hydride in toluene (1.8 mL, 2.7mmol) was added to the solution 1.05 g (2.06 mmol) of 38 in 10 mL of THFat −78° C. After 1 h, 4 mL of methanol was added and the mixture waswarmed to 25° C., then poured into 40 mL of ethyl acetate/saturatedaqueous potassium sodium tartrate (1/1). Layers were separated and theaqueous phase was further extracted with ethyl acetate (3×30 mL). Thecombined organic layers were then dried over MgSO₄, filtered,concentrated, and the residue was chromatographed on silica gel (ethylacetate) to afford 740 mg (70%) of lactol 39.

A 1.5M solution of potassium t-butoxide in THF (8.6 mL, 8.6 mmol) wasadded dropwise to a mixture of 15 mL of THF and 1.92 g (4.33 mmol) ofphosphonium salt 29 at 0° C. After stirring for 1 h, a solution of 740mg (1.45 mmol) of lactol 39 in 5 mL of THF was added dropwise, and thereaction was allowed to warm to 25° C. overnight. The mixture was thenpoured into 100 mL of ethyl acetate/saturated NH₄Cl (1/1). Layers wereseparated, and the aqueous phase was further extracted with ethylacetate (2×70 mL). Combined organic layers were dried over MgSO₄,filtered, and concentrated to afford 1.6 g of crude acid 40.

Crude acid 40 (1.6 g) was dissolved in 11 mL of acetone and cooled to 0°C., then 850 mg (5.6 mmol) of DBU was added dropwise to the solution.The resulting mixture was stirred for 15 min at 0° C. and 30 min at 25°C., after which 850 mg (5.0 mmol) of isopropyl iodide was added. Thereaction was stirred overnight and poured into 100 mL of ethylacetate/saturated NH₄Cl (1/1). Layers were separated, and the aqueousphase was further extracted with ethyl acetate (2×50 mL). Combinedorganic layers were dried over MgSO₄, filtered and concentrated. Theresulting residue was purified by silica gel chromatography (ethylacetate/hexanes, 3/2) to afford 560 mg (61% from lactol 39) of isopropylester 41.

D: (5Z)-(9S, 11R, 15R)-16-(3-Chlorophenoxy)-17,18,19,20-tetranor-9,11,15-trihydroxy-5-prostenolPivaloate (8)

A solution of 400 mg (0.63 mmol) of 41 in 5 mL of THF was added dropwiseto a suspension of 35 mg (0.92 mmol) of lithium aluminum hydride in 5 mLof THF at 0° C. After 2 h, the reaction was poured into 50 mL of a 1/1mixture of ethyl acetate/saturated NaHCO₃. The layers were thenseparated, and the aqueous phase was extracted with ethyl acetate (2×2mL). Combined organic layers were dried over MgSO₄, filtered, andconcentrated. The resulting residue was purified by silica gelchromatography (ethyl acetate) to afford 350 mg (95%) of diol 42.

Pivaloyl chloride (90 mg, 0.75 mmol) was added to a mixture of 350 mg(0.60 mmol) of 42, 60 mg (0.76 mmol) of pyridine, 22 mg (0.18 mmol) of4-(dimethylamino)pyridine, and 7 mL of CH₂Cl₂. After 1.5 h, the mixturewas poured into 30 mL of saturated NH₄Cl/ethyl acetate (1/1). Layerswere then separated and the aqueous phase was extracted with ethylacetate (2×20 mL). The combined organic layers were dried over MgSO₄,filtered, concentrated, and purified by silica gel chromatography (ethylacetate/hexane, 3/2) to afford 370 mg (93%) of pivaloate 43.

Water (approximately 10 drops) and concentrated HCl (approximately 3drops) were added to a solution of 370 mg (0.56 mmol) of 43 in 5 mL ofmethanol. After stirring overnight, the reaction was quenched by theaddition of 20 mL of saturated NaHCO₃, and the mixture was extractedwith ethyl acetate (3×20 mL). The combined organic layers were driedover MgSO₄, filtered, and concentrated. The residue was chromatographedon silica gel (ethyl acetate/hexane, 3/2), to afford 165 mg (59%) oftriol 8. ¹³C NMR (CDCl₃) δ 178.77 (C), 159.27 (C), 134.80 (C), 130.20(CH), 128.62 (CH), 121.19 (CH), 114.97 (CH), 112.97 (CH), 78.50 (CH),74.46 (CH), 72.31 (CH₂), 69.86 (CH), 64.16 (CH₂), 52.53 (CH), 51.67(CH), 42.50 (CH₂), 31.51 (CH₂), 29.40 (CH₂), 28.10 (CH₂), 27.12 (CH₃),26.77 (CH₂), 26.65 (CH₂), 25.77 (CH₂). Cl MS, m/z calcd for C₂₇H₄₁O₆Cl₁(MH⁺), 497.2670, found 497.2656.

Included within the scope of the present invention are the individualenantiomers of the title compounds, as well as their racemic andnon-racemic mixtures. The individual enantiomers can beenantioselectively synthesized from the appropriate enantiomericallypure or enriched starting material by means such as those describedbelow. Alternatively, they may be enantioselectively synthesized fromracemic/non-racemic or achiral starting materials. (Asymmetric Synthesisby J. D. Morrison and J. W. Scott, Ed., Academic Press Publishers: NewYork, 1983-1985 (five volumes published over a three year span withchapters contributed by about two dozen authors) and Principles ofAsymmetric Synthesis by R. E. Gawley and J. Aube, Ed., ElsevierPublishers: Amsterdam, 1996). They may also be isolated from racemic andnon-racemic mixtures by a number of known methods, e.g. by purificationof a sample by chiral HPLC (A Practical Guide to Chiral Separations byHPLC, G. Subramanian, Ed., VCH Publishers: New York, 1994; ChiralSeparations by HPLC, A.M. Krstulovic, Ed., Ellis Horwood Ltd.Publishers, 1989), or by enantioselective hydrolysis of a carboxylicacid ester sample by an enzyme (Ohno, M.; Otsuka, M. Organic Reactions,volume 37, page 1 (1989)). Those skilled in the art will appreciate thatracemic and non-racemic mixtures may be obtained by several means,including without limitation, nonenantioselective synthesis, partialresolution or even mixing samples having different enantiomeric ratios.Also included within the scope of the present invention are theindividual isomers substantially free of their respective enantiomers.

EXAMPLE 5

PGF_(2α) analogues are known to contract the iris sphincter of cats andthis assay is a generally accepted reference for activity. For thisreason, the pupil diameter of cats may be used to define the activity ofPGF_(2α) analogues and, as demonstrated by Stjernschantz and Resul(Drugs Future, 17:691-704 (1992)), predict the lOP-lowering potency.

Compounds of the present invention were therefore screened for pupillaryconstriction in the cat. Data for compounds 6, 7, and 8 are presented inTable 3, below. The response is quantitated as Area ₁₋₅ values (areaunder the pupil diameter versus time curve from 1-5 hours), and theequivalent response dose (ED₅) is estimated from its dose responserelationship.

TABLE 3 Cat Pupil Diameter Response Compound ED₅ (μg) PGF_(2α) IsopropylEster 0.02 Cloprostenol Isopropyl Ester 0.01 6 0.2  7 0.02 8 0.06

Discussion

The two standard compounds, PGF_(2α) isopropyl ester and cloprostenolisopropyl ester, produced marked change in cat pupillary diameter,displaying ED₅ values of 0.02 and 0.01 μg, respectively. Compound 7(cloprostenol-1-ol) and compound 8 (13,14-dihydrocloprostenol-1-olpivaloate), displayed nearly equivalent potency.13,14-Dihydrofluprostenol isopropyl ester (compound 6) was approximatelyone order of magnitude less potent, with an ED₅ of 0.2 μg.

EXAMPLE 6

In the study presented below, compound 6 (Table 2, above) was tested forlOP-lowering effect in cynomolgus monkey eyes.

The right eyes of the cynomolgus monkeys used in this study werepreviously given laser trabeculoplasty to induce ocular hypertension inthe lasered eye. Animals had been trained to sit in restraint chairs andconditioned to accept experimental procedures without chemicalrestraint. lOP was determined with a pneumatonometer after light cornealanesthesia with dilute proparacaine. The test protocol included afive-dose treatment regimen because of the typical delayed response toprostaglandins. The designated test formulations were administered tothe lasered right eyes, and the normal left eyes remained untreated,although lOP measurements were taken. Baseline lOP values weredetermined prior to treatment with the test formulation, and then lOPwas determined from 1 to 7 hours after the first dose, 16 hours afterthe fourth dose, and 1 to 4 hours after the fifth dose.

The equivalent response dose (ED₂₀) is estimated from the dose responserelationship to be the dose producing a 20% peak reduction in lOP.

TABLE 4 Monkey IOP Response Compound ED₂₀ (ig) PGF_(2α) Isopropyl Ester0.4 6 0.3

Discussion

As can be seen in Table 4, compound 6, the 13,14-dihydro analogue offluprostenol was quite potent in the monkey lOP model, producing a 20%reduction at 0.3 μg. This was even more potent than the standardcompound, PGF_(2α) isopropyl ester.

EXAMPLE 7

Improvement of Optic Nerve Head Blood Flow after One-Week TopicalTreatment with Compound 1B in the Rabbit.

Purpose

A two-way crossover study design was used to compare the effects ofCompound 1B (0.004%), topically applied, and dosing vehicle onmicrovascular optic nerve head blood flow, blood pressure, heart rate,and acid-base balance in fifteen acepromazine-tranquilized Dutch-Beltedrabbits.

Methods

Baseline measurements were taken before treatment and after drug-freewashout periods of 14 days. Microvascular optic nerve head (ONH) bloodflow was measured with a fundus camera-based Laser Doppler flowmeter(LDf). Thirty microliters o a 0.004% solution of Compound 1B or vehiclewas administered in left eyes only q.d. for 7 days. Experimentealmeasurements wre made 2 hours after the topical dose was administered onday 8. A sterile 23 gauge intracatheter was inserted into the centralear artery to monitor blood pressure and to take blood samples for bloodpH/gas analysis.

Results

ONH blood flow was significantly increased (p<0.05) in rabbits treatedwith Compound 1B, when compared to the vehicle treated group. The mean±SEM percent increase from baseline was 13.4±3.9 in Compound 1B treatedanimals and −1.0±4.3 in vehicle treated animals. Only smallnon-significant changes in systemic blood pressure, heart rate, bloodgas tensions and pH were present in the Compound 1B treatment group whencompared with the vehicle group. The observed mean percent decreases insystemic arterial pH, and pO₂ and arterial PCO₂ tensions were 0.07±0.09,1.44±2.03, and 2.59±2.5, respectively.

Conclusion

One week of once a day topical ocular treatment with Compound 1Bsignificantly increased ONH blood flow in tranquilized Dutch-Beltedrabbits, while eliciting minimal systemic acid-base balancedisturbances. This suggests that the mechanism responsible for thepositive effect on the ONH vasculature involves a local ocular actionsince the blood flow increase in this study occurred without significantchanges in systemic blood pressure or arterial blood gases/pH.

EXAMPLE 8

The following Formulations 14 are representative pharmaceuticalcompositions of the invention for topical use in lowering of intraocularpressure. Each of Formulations 1 through 4 may be formulated inaccordance with procedures known to those skilled in the art.

FORMULATION 1 Ingredient Amount (wt %) Compound 5 (Table 2) 0.002Dextran 70 0.1 Hydroxypropyl methylcellulose 0.3 Sodium chloride 0.77Potassium chloride 0.12 Disodium EDTA 0.05 Benzalkonium chloride 0.01HCl and/or NaOH pH 7.2-7.5 Purified water q.s. to 100%

FORMULATION 2 Ingredient Amount (wt %) Compound 6 (Table 2) 0.01Monobasic sodium phosphate 0.05 Dibasic sodium phosphate 0.15(anhydrous) Sodium chloride 0.75 Disodium EDTA 0.01 Benzalkoniumchloride 0.02 Polysorbate 80 0.15 HCl and/or NaOH pH 7.3-7.4 Purifiedwater q.s. to 100%

FORMULATION 3 Ingredient Amount (wt %) Compound 7 (Table 2) 0.001Dextran 70 0.1 Hydroxypropyl methylcellulose 0.5 Monobasic sodiumphosphate 0.05 Dibasic sodium phosphate 0.15 (anhydrous) Sodium chloride0.75 Disodium EDTA 0.05 Benzalkonium chloride 0.01 NaOH and/or HCl pH7.3-7.4 Purified water q.s. to 100%

FORMULATION 4 Ingredient Amount (wt %) Compound 8 (Table 2) 0.003Monobasic sodium phosphate 0.05 Dibasic sodium phosphate 0.15(anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05 Benzalkoniumchloride 0.01 HCl and/or NaOH pH 7.3-7.4 Purified water q.s. to 100%

The invention has been described by reference to certain preferredembodiments; however, it should be understood that it may be embodied inother specific forms or variations thereof without departing from itsspirit or essential characteristics. The embodiments described above aretherefore considered to be illustrative in all respects and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than by the foregoing description.

What is claimed is:
 1. A method of treating glaucoma and ocularhypertension which comprises topically administering to the affected eyea composition comprising a therapeutically effective amount of acombination of a first compound selected from the group consisting ofbeta-blockers, carbonic anhydrase inhibitors, adrenergic agonists, andcholinergic agonists; together with a second compound having theabsolute stereochemical structure of the following formula (IV):

wherein: R₁=H; C₁-C₁₂ straight-chain or branched alkyl; C₁-C₁₂straight-chain or branched acyl; C₃-C₈ cycloalkyl; or a cationic saltmoiety; R₂, R₃=H, or C₁-C₅ straight-chain or branched alkyl; or R₂ andR₃ taken together may represent O; X=O, S, or CH₂; - - - represents anycombination of a single bond, or a cis or trans double bond for thealpha (upper) chain; and a single bond or trans double bond for theomega (lower) chain; R₉=H, C₁-C₁₀ straight-chain or branched alkyl, orC₁-C₁₀ straight-chain or branched acyl; R₁₁=H, C₁-C₁₀ straight-chain orbranched alkyl, or C₁-C₁₀ straight-chain or branched acyl; Y=O; or H andOR₁₅ in either configuration wherein R₁₅=H, C₁—C₁₀ straight-chain orbranched alkyl, or C₁-C₁₀ straight-chain or branched acyl; and Z=Cl orCF₃; with the proviso that when R₂ and R₃ taken together represent O,then R₁≠C₁-C₁₂ straight-chain or branched acyl; and when R₂=R₃=H, thenR₁≠a cationic salt moiety.
 2. The method of claim 1, wherein for thecompound (IV): R₂, R₃ taken together represent O; X=CH₂; - - -represents a cis double bond for the alpha (upper) chain and a transdouble bond for the omega (lower) chain; R₉ and R₁₁=H; and Y=OH in thealpha configuration and H in the beta configuration.
 3. The method ofclaim 2, wherein for the compound (IV): Z=CF₃.
 4. The method of claim 1,wherein: R₂=R₃=H, or R₂ and R₃ taken together represent O; X=O or CH₂;R₉=R₁₁=H; Y=H and OR₁₅; and R₁₅=H.
 5. The method of claim 4, wherein:R₁=H, C₁-C₁₂ straight chain or branched alkyl or cationic salt moiety;and R₂ and R₃ taken together represent O.
 6. The method of claim 5,wherein the compound of formula (IV) is selected from the groupconsisting of cloprostenol, fluprostenol, 3-oxacloprostenol,13,14-dihydrofluprostenol, and their pharmaceutically acceptable estersand salts.
 7. The method of claim 4, wherein the first compound isbeta-blocker.
 8. The method of claim 7, wherein the beta-blocker isselected from the group consisting of timolol, betaxolol andlevobetaxolol.
 9. The method of claim 8, wherein the beta-blocker istimolol and the compound of formula (IV) is fluprostenol isopropylester.
 10. The method of claim 4, wherein the first compound is anadrenergic agonist.
 11. The method of claim 10, wherein the adrenergicagonist is selected from the group consisting of apraclonidine andbrimonidine.
 12. The method of claim 11, wherein the adrenergic agonistis brimonidine and the compound of formula (IV) is fluprostenolisopropyl ester.
 13. The method of claim 1, wherein between about 0.01and about 1000 μg/eye of the compounds of formula (IV) is administered.14. The method of claim 13, wherein between about 0.1 and about 100μg/eye of the compound of formula (IV) is administered.
 15. The methodof claim 14, wherein between about 0.1 and about 10 μg/eye of thecompound of formula (IV) is administered.
 16. A topical ophthalmiccomposition for the treatment of glaucoma and ocular hypertensioncomprising an ophthalmically acceptable carrier and a therapeuticallyeffective amount of a combination of a first compound selected from thegroup consisting of beta-blockers, carbonic anhydrase inhibitors,adrenergic agonists, and cholinergic agonists; together with a secondcompound having the absolute stereochemical structure of the followingformula (IV) and being substantially free of the enantiomer of saidcompound:

wherein: R₁=H; C₁-C₁₂ straight-chain or branched alkyl; C₁-C₁₂straight-chain or branched acyl; C₃-C₈ cycloalkyl; or a cationic saltmoiety; R₂, R₃=H, or C₁-C₅ straight-chain or branched alkyl; or R₂ andR₃ taken together may represent O; X=O, S, or CH₂; - - - represents anycombination of a single bond, or a cis or trans double bond for thealpha (upper) chain; and a single bond or trans double bond for theomega (lower) chain; R₉=H, C₁-C₁₀ straight-chain or branched alkyl, orC₁-C₁₀ straight-chain or branched acyl; R₁₁=H, C₁-C₁₀ straight-chain orbranched alkyl, or C₁-C₁₀ straight-chain or branched acyl; Y=O; or H andOR₁₅ in either configuration wherein R₁₅=H, C₁-C₁₀ straight-chain orbranched alkyl, or C₁-C₁₀ straight-chain or branched acyl; and Z=Cl orCF₃; with the proviso that when R₂ and R₃ taken together represent O,then R₁≠C₁-C₁₂ straight-chain or branched acyl; and when R₂=R₃=H, thenR₁≠a cationic salt moiety; and with the further proviso that thefollowing compound be excluded: cyclopentaneheptenol-5-cis-2-(3-αhydroxy-4-m-chlorophenoxy-1-trans-butenyl)-3,5dihydroxy, [1_(α), 2_(β), 3_(α), 5_(α)].
 17. The composition of claim16, wherein for the compound (IV): R₂, R₃ taken together represent O;X=CH₂; - - - represents a cis double bond for the alpha (upper) chainand a trans double bond for the omega (lower) chain; R₉ and R₁₁=H; andY=OH in the alpha configuration and H in the beta configuration.
 18. Thecomposition of claim 17, wherein for the compound (IV): Z=CF₃.
 19. Amethod of treating an optic nerve disorder, which comprisesadministering to the affected eye a composition comprising atherapeutically effective amount of a compound having the absolutestereochemical structure of the following formula (IV) an beingsubstantially free of the enantiomer of said compound:

wherein: R₁=H; C₁-C₁₂ straight-chain or branched alkyl; C₁-C₁₂straight-chain or branched acyl; C₃-C₈ cycloalkyl; or a cationic saltmoiety; R₂, R₃=H, or C₁-C₅ straight-chain or branched alkyl; or R₂ andR₃ taken together may represent O; X=O, S, or CH₂; - - - represents anycombination of a single bond, or a cis or trans double bond for thealpha (upper) chain; and a single bond or trans double bond for theomega (lower) chain; R₉=H, C₁-C₁₀ straight-chain or branched alkyl, orC₁-C₁₀ straight-chain or branched acyl; R₁₁=H, C₁-C₁₀ straight-chain orbranched alkyl, or C₁-C₁₀ straight-chain or branched acyl; Y=O; or H andOR₁₅ in either configuration wherein R₁₅=H, C₁-C₁₀ straight-chain orbranched alkyl, or C₁-C₁₀ straight-chain or branched acyl; and Z=Cl orCF₃; with the proviso that when R₂ and R₃ taken together represent O,then R₁≠C₁-C₁₂ straight-chain or branched acyl; and when R₂=R₃=H, thenR₁≠a cationic salt moiety; and with the further proviso that thefollowing compound be excluded: cyclopentaneheptenol-5-cis-2-(3-αhydroxy-4-m-chlorophenoxy-1-trans-butenyl)-3,5dihydroxy, [1_(α), 2_(β), 3_(α), 5_(α)].
 20. The method of claim 19,wherein the compound of formula (IV) is fluprostenol and itspharmaceutically acceptable esters and salts.
 21. The method of claim20, wherein the compound of formula (IV) is fluprostenol isopropylester.